Wound dressing and a method for producing the same

ABSTRACT

The invention provides a wound dressing made by an ex vivo formed combination of fibrinogen and/or fibrin containing-liquid formulation and an oxidized cellulose (OC) backing; and use thereof.

TECHNOLOGICAL FIELD

The present disclosure relates, inter alia, to therapeutic devices andmethods for treating wounds.

BACKGROUND

References considered to be relevant as background to the presentlydisclosed subject matter are listed below.

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

The control of bleeding is essential and critical in surgical proceduresto minimize blood loss, to reduce post-surgical complications, and toshorten the duration of the surgery in the operating room. TopicalAbsorbable Hemostats (TAH) are one option for managing and controllingbleeding that are widely used in surgical applications.

Relevant prior art references are: Ishii, Keiichi, et al. Journal ofEndourology, 25(11), 1775-1779 (2011); Ishii, Keiichi, et al.International Journal of Urology 18(6), 478-482 (2011), Lewis, K. M., etal. European Surgery 45(4), 213-220 (2013); Spangler, Daniel et al.Surgical Infections 4(3), 255-262 (2003); and DeAnglis, Ashley P. et al.Blood Coagulation & Fibrinolysis 28, 134-138 (2017).

US Patent application, publication No. 2012/0209319, describes areinforced multi component adhesive comprising layer of uncured andcurable composition of matter, and a biocompatible inert reinforcingagent comprising at least one curing agent and a thin film, e.g.oxidized regenerated cellulose.

GENERAL DESCRIPTION

The present disclosure provides, in accordance with a first of itsaspects, a method for producing a wound dressing to be applied to atissue comprising a liquid formulation comprising fibrinogen and/orfibrin (the liquid formulation is also referred to as:

“fibrinogen and/or fibrin-containing liquid formulation”,interchangeably) with an oxidized cellulose (OC) backing which are exvivo combined to thereby obtain the wound dressing.

According to an aspect of the present invention, there is provided amethod for producing wound dressing to be applied onto a tissue, themethod comprising ex vivo combining a liquid formulation comprisingfibrinogen and/or fibrin with an oxidized cellulose (OC) backing tothereby obtain the wound dressing.

In some embodiments of the method, the OC backing has a wound facingside and the liquid formulation is combined at least with the woundfacing side.

In some embodiments of the method, the wound dressing is to be appliedto a tissue within less than 20 second from the combining.

In some embodiments of the method, the OC backing comprises oxidizedregenerated cellulose (ORC).

In some embodiments of the method, the OC backing is in the form of aknitted, woven or non-woven fabric.

In some embodiments of the method, the liquid formulation comprisesthrombin.

In some embodiments of the method, the combining comprises any one of:spraying, smearing, brushing, casting, printing, injecting and soakingthe liquid formulation.

In some embodiments of the method, the thrombin is present at aconcentration ranging from 400 to 1200 IU/ml.

In some embodiments of the method, the fibrinogen and/or fibrin arepresent at a concentration ranging from 10 to 120 mg/ml.

According to a further aspect of the present invention, there isprovided a wound dressing comprising an ex vivo formed combination of aliquid formulation comprising fibrinogen and/or fibrin (i.e. fibrinogenand/or fibrin containing-liquid formulation) and an oxidized cellulose(OC) backing.

In some embodiments of the wound dressing, the liquid formulation iscombined at least partially with a wound facing side of the wounddressing.

In some embodiments of the wound dressing, the OC backing comprisesoxidized regenerated cellulose (ORC).

In some embodiments of the wound dressing, the OC backing is in the formof a knitted, woven or non-woven fabric.

In some embodiments of the wound dressing, the liquid formulationfurther comprises thrombin.

In some embodiments of the wound dressing, the thrombin is present at aconcentration ranging from 400 to 1200 IU/ml.

In some embodiments of the wound dressing, the fibrinogen and/or fibrinare present at a concentration ranging from 10 to 120 mg/ml.

In some embodiments of the wound dressing, the liquid formulation issprayed, smeared, brushed, casted, printed or soaked within the backing.

In the relevant context of the present disclosure, when referring to“combined”, or any grammatical inflection thereof, it is to beunderstood as any form of association/contacting between the liquidformulation and OC backing. This may include, without being limitedthereto, any one or combination of spraying, smearing, dipping, soaking,brushing, casting, printing, injecting, coating, as further describedbelow.

In accordance with yet another aspect, there is also provided by thepresent disclosure, a method of treating (also referred to as: “thetreatment method”) a wounded tissue in a subject, the method comprising:

(a) ex vivo combining a liquid formulation comprising fibrinogen and/orfibrin with an oxidized cellulose (OC) backing to form a wound dressing;and

(b) applying the wound dressing onto a wound, the applying being carriedout within less than 20 seconds from the combining.

In some embodiments of the treatment method, the OC backing has a woundfacing side and the liquid formulation is combined at least with thewound facing side, wherein the wound facing side is applied onto thewounded tissue.

In some embodiments of the treatment method, the wounded tissue is ableeding tissue.

In some embodiments of the treatment method, the backing comprisesoxidized regenerated cellulose (ORC).

In some embodiments of the treatment method, the OC backing is in theform of a knitted, woven or non-woven fabric.

In some embodiments of the treatment method, the formulation furthercomprises thrombin.

In some embodiments of the treatment method, the thrombin is present ata concentration ranging from 400 to 1200 IU/ml.

In some embodiments of the treatment method, the fibrinogen and/orfibrin are present at a concentration ranging from 10 to 120 mg/ml.

In some embodiments of the treatment method, the combining comprises anyone of spraying, smearing, brushing, casting, printing, injecting andsoaking of the liquid formulation.

In some embodiments of the treatment method, the method furthercomprises pressing the wound dressing against the wounded tissue.

In some embodiments of the treatment method, the pressing is carried outfor a time sufficient to allow adherence of the wound dressing to atleast a portion of the wounded tissue.

In some embodiments of the treatment method, the subject is a humansubject.

In some embodiments of the treatment method, the method is for treatinga wound during a surgical procedure.

In some embodiments of the treatment method, the surgical procedurecomprises abdominal surgery, cardiovascular surgery, thoracic surgery,head and neck surgery, pelvic surgery, skin and subcutaneous tissueprocedure.

In some embodiments of the treatment method, the wounded tissuecomprises a bleeding artery.

In some embodiments of the treatment method, the treatment comprisesreducing bleeding.

In accordance with a further aspect, there is provided by the presentdisclosure a kit for treating a wounded tissue in a subject comprising:a first container comprising an oxidized cellulose (OC) backing; and asecond container comprising a formulation comprising fibrinogen and/orfibrin.

In some embodiments of the kit, the formulation is in a liquid or in afrozen form.

In some embodiments of the kit, the kit comprises instructions for exvivo use of the OC backing in combination with the formulation.

In some embodiments of the kit, the OC backing comprises oxidizedregenerated cellulose (ORC).

In some embodiments of the kit, the OC backing is in the form of aknitted, woven or non-woven fabric.

In some embodiments of the kit, the kit comprises thrombin within thesecond container.

In some embodiments of the kit, the thrombin is present at aconcentration ranging from 400 to 1200 IU/ml.

In some embodiments of the kit, the fibrinogen and/or fibrin are presentat a concentration ranging from 10 to 120 mg/ml.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWING

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of a non-limiting exampleonly, with reference to the accompanying drawing, in which:

FIG. 1 is a bar graph showing the adhesion intensity of a wound dressingprepared ex vivo with crosslinking (curing) times of around 0(immediately) and after 20 seconds. For each crosslinking time,right-hand bars (vertical stripes) represent Evicel® drip on padpreparation according to the invention, while the left-hand bars(horizontal stripes) represent in situ preparation of Evicel® dripdirectly on tissue, as described in Example 1. The right-most bar(diagonal stripes, only Surgicel) represents ORC only. Error barsrepresent standard deviation.

DETAILED DESCRIPTION OF EMBODIMENTS

During surgical procedures, a surgeon must be able to addresshomeostasis.

Topical Absorbable Hemostats (TAH) encompass products based on oxidizedcellulose (OC), oxidized regenerated cellulose (ORC), gelatin, collagen,chitin, chitosan, etc. To improve the hemostatic performance, scaffoldsbased on the above materials can be combined with biologically-derivedclotting factors, such as, and without being limited thereto, thrombinand fibrinogen.

The present disclosure is based on the realization that in many cases,especially when the bleeding in the wound is severe, when applying abiological glue, e.g. liquid fibrin/fibrinogen-based sealant, directlyonto a bleeding tissue, the glue might be washed away or diluted by theflow of the blood and thus, be less effective in reducing the bleeding.

An object of the present disclosure is to overcome the drawback ofco-administration of oxidized cellulose and thrombin and/or fibrinogenin view of the acidity of the oxidized cellulose. Such acidityimmediately denatures plasma proteins (e.g., thrombin, fibrinogen,tissue factor, etc.). Therefore, co-administration of oxidized celluloseand thrombin, fibrinogen, or alike, might render these plasma proteinsineffective.

It was found according to the disclosure that, surprisingly, when theliquid sealant matter and specific kinds of TAH, such as ORC, arecombined ex vivo, the bleeding rate is beneficially reduced,particularly if placed onto the wound within a specified time windowfrom the combination of the same.

Herein, by “combined ex vivo” (or “ex vivo combined”) it is meant thatthe step of combining the liquid sealant matter and the specific kind ofTAH, such as ORC, is not employed upon a physical contact of either thesealant matter or the specific kind of TAH (e.g., ORC) with the bleedingsite.

Accordingly, the present disclosure provides, in one aspect thereof, amethod for producing a wound dressing to be applied to a tissue, themethod comprising ex vivo combining a fibrinogen and/orfibrin-containing liquid formulation with an oxidized cellulose (OC)backing, such as, and without being limited thereto, an ORC backing, tothereby obtain the wound dressing.

In one such embodiment, the backing has a wound facing side surface andthe liquid formulation is combined, at least partially with the woundfacing side surface. Hereinthroughout, by “combined, at least partiallywith the wound facing side surface” it is meant that at least a portionof a surface of the wound facing side is combined with the liquidformulation.

In some such embodiments, the liquid formulation is in the form of alayer on the surface.

The thickness of the patch (e.g., pad) may be about 1 mm or above, suchas about 45 mm, or, in some embodiments, may be about 1 to about 45 mm.

Accordingly, in some embodiments, the thickness of patch (e.g., pad) is1, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, or 45 mm, including any value andrange therebetween.

In some embodiments, the term “at least a portion of the surface of thewound facing side” refers to at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or even 100% of the surface of the wound facingside.

In one embodiment, the wound dressing is to be applied or is configuredbe applied to a tissue within less than 20 second from the ex vivocombining.

In some embodiments, application of the wound dressing may be performedwithin 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 5, 4, 3, 2, or1 seconds or even immediately upon the combining. In some embodiments,the application onto the wound is carried out after at least 2 to 5seconds from combining, or in some embodiments, after at least 2 to 10seconds from combining.

The term “immediately” refers to around 0 seconds, for example from 0-2seconds.

In some embodiments, the disclosed wound dressing is configured to beapplied onto a tissue within 2 to 20 seconds from the combining. In somesuch embodiments, as further described in the Examples section below,the ex vivo combining of the liquid formulation with an OC (e.g., ORC)backing, provides a wound dressing characterized by adherence strengthof at least 50% or at least 60%, e.g., at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%, compared to the correspondingadherence strength provided upon in-situ preparation of fibrin sealantdrip directly on the bleeding tissue and then combining with the OC(e.g., ORC).

In some embodiments, the ex vivo combining of the liquid formulationwith an OC (e.g., ORC) backing, provides a wound dressing characterizedby adherence strength of 50 to 90% compared to the correspondingadherence strength provided upon in-situ preparation of fibrin sealantdrip directly on the bleeding tissue and then combining with the OC(e.g., ORC).

Herein, the term “adhesion strength” refers to the pull-force strengthrequired to break the contact between the wound dressing and the tissue.

In a further embodiment, the backing comprises ORC.

In a further embodiment, the backing comprises ORC having an oxidationlevel ranging from 12 to 21%. In a further embodiment, the backingcomprises ORC having an oxidation level ranging from 18 to 21%.

In a further embodiment, the backing comprises ORC having an oxidationlevel of 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21%, including any valueand range therebetween.

In some embodiments, the oxidation level refers to the carboxyl contentof the ORC.

Methods for determining the oxidation level are known in the art. Inexemplary embodiments, the oxidation level is determined per UnitedStates Pharmacopeia (USP) 23-NF18.

In a further embodiment, the backing is in the form of a knitted fabric.

In some embodiments, the backing is in the form of woven or non-wovenfabric.

In a further embodiment, the formulation comprises thrombin.

In a further embodiment, the sealant formulation comprises atwo-component formulation comprising: a thrombin component, such as, andwithout being limited thereto, a liquid thrombin component, and afibrinogen component, such as, and without being limited thereto, aliquid fibrinogen component.

In a further embodiment, the sealant formulation comprises atwo-component formulation comprising: a thrombin component, such as, andwithout being limited thereto, a liquid thrombin component, and a fibrincomponent, such as, and without being limited thereto, a liquid fibrincomponent.

In a further embodiment, the sealant formulation comprises a fibrincomponent, such as, and without being limited thereto, a liquid fibrincomponent.

In a further embodiment, the sealant formulation comprises atwo-component formulation comprising: a thrombin component, such as, andwithout being limited thereto, a liquid thrombin component; and acomponent comprising fibrin and fibrinogen, such as, and without beinglimited thereto, a liquid fibrin-and-fibrinogen component.

In a further embodiment of the sealant formulation, the thrombin ispresent at a concentration of about 2 to 1200 IU/ml, 2.5 to 1200 IU/ml,8 to 1200 IU/ml, 80 to 1200 IU/ml, 400 to 1200 IU/ml, 400 to 600 IU/ml,or 800 to 1200 IU/ml.

In a further embodiment of the sealant formulation, the thrombin ispresent at a concentration of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 10, 50, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, or 1200 IU/ml, including any value and range therebetween.

In a further embodiment of the formulation, the thrombin is present atthe thrombin component at concentration of about 2 to 1200 IU/ml, 2.5 to1200 IU/ml, 8 to 1200 IU/ml, 80 to 1200 IU/ml, 400 to 1200 IU/ml, 400 to600 IU/ml, or 800 to 1200 IU/ml.

In a further embodiment of the formulation, the thrombin is present atthe thrombin component at a concentration of about 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 10, 50, 80, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, or 1200 IU/ml, including any value andrange therebetween.

As used herein, the term “IU” denotes “International Units” and may bedetermined by the clotting assay against an internal reference standardfor potency concentration measurement that has been calibrated against,for example, the World Health Organization (WHO) Second InternationalStandard for Thrombin, 01/580. A unit (U) is equivalent to anInternational Unit (IU).

In a further embodiment of the sealant formulation, the fibrinogen ispresent at a concentration of about 10 to 120 mg/ml, 10 to 105 mg/ml, 25to 90 mg/ml, 30 to 45 mg/ml, 60 to 90 mg/ml, 60 to 110 mg/ml, or 55 to85 mg/ml.

In a further embodiment of the sealant formulation, the fibrinogen ispresent at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 mg/ml,including any value and range therebetween.

In a further embodiment of the formulation, the fibrinogen is present atthe fibrinogen component at a concentration of about 10 to 120 mg/ml, 10to 105 mg/ml, 25 to 90 mg/ml, 30 to 45 mg/ml, 60 to 90 mg/ml, 60 to 110mg/ml, or 55 to 85 mg/ml.

In a further embodiment of the formulation, the fibrinogen is present atthe fibrinogen component at a concentration of 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or120 mg/ml, including any value and range therebetween.

In some embodiments, the two components of the formulation (i.e.thrombin and fibrinogen) are combined in a ratio (v/v) ranging fromabout 1.5:1 to about 1:1.5. In some embodiments, the two components ofthe formulation (i.e. thrombin and fibrinogen) are combined in a ratio(v/v) ranging from about 1.2:1 to about 1:1.2.

In exemplary embodiments the two components of the formulation (i.e.thrombin and fibrinogen) are combined in a ratio (v/v) of about 1:1.

In a further embodiment of the sealant formulation, the fibrin ispresent at a concentration of about 10 to 120 mg/ml, 10 to 105 mg/ml, 25to 90 mg/ml, 30 to 45 mg/ml, 60 to 90 mg/ml, 60 to 110 mg/ml, or 55 to85 mg/ml.

In a further embodiment of the sealant formulation, the fibrin ispresent at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 mg/ml,including any value and range therebetween.

In a further embodiment of the formulation, the combination offibrinogen and fibrin is present at a concentration of about 10 to 120mg/ml, 10 to 105 mg/ml, 25 to 90 mg/ml, 30 to 45 mg/ml, 60 to 90 mg/ml,60 to 110 mg/ml, or 55 to 85 mg/ml.

In a further embodiment of the formulation, the combination offibrinogen and fibrin is present at a concentration of 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, or 120 mg/ml, including any value and range therebetween.

In a further embodiment, the combining comprises one or more stepsselected from: spraying, smearing, brushing, casting, printing,injecting and soaking the fibrinogen and/or fibrin containing liquidformulation.

Also provided by the present disclosure is:

a wound dressing comprising an ex vivo formed combination of fibrinogenand/or fibrin containing-liquid formulation and an oxidized cellulose(OC) backing, such as, and without being limited thereto, an ORCbacking.

In one such embodiment, the backing has a wound facing side and theliquid formulation is combined, at least partially, with the woundfacing side. In another embodiment, the wound dressing is to be appliedto a tissue within less than 20 second from the combining. In a furtherembodiment, the backing is in the form of a knitted, woven or non-wovenfabric.

In a further embodiment, the formulation comprises thrombin.

According to another aspect of the present invention, there is provideda method of treating a wounded tissue in a subject, the methodcomprising (a) ex vivo combining a fibrinogen and/or fibrin containingliquid formulation with a topical absorbable hemostat (TAH) backing or ascaffold comprising OC, such as, without limitation, ORC, to form awound dressing; and (b) applying the wound dressing onto a wound, theapplication being within less than 20 seconds from the combining.

In one such embodiment of the treating method, the backing or scaffoldhas a wound facing side and the liquid formulation is combined at leastpartially with the wound facing side.

In one embodiment, the wounded tissue is a bleeding tissue.

In a further embodiment, the backing comprises ORC.

In a further embodiment, the backing is in the form of a knitted, wovenor non-woven fabric.

In a further embodiment, the fibrinogen and/or fibrin containingformulation further comprises thrombin.

In one embodiment the formulation comprises a two-component formulationcomprising: a thrombin component, such as, and without being limitedthereto, a liquid thrombin component, and a fibrinogen component, suchas, and without being limited thereto, a liquid fibrinogen component.

In one such embodiment, the combining comprises one or more stepsselected from spraying, smearing, brushing, casting, printing, injectingand soaking the fibrinogen and/or fibrin containing liquid formulation

In one embodiment, following the combining, the method comprises a stepof pressing the wound dressing against the wounded tissue.

In a further embodiment, the pressing is carried out for a timesufficient to allow adherence of the wound dressing to at least aportion of the wounded tissue.

In a further embodiment, the method is for treating a wound during asurgical procedure.

In a further embodiment, the surgical procedure comprises a procedureselected from abdominal surgery, cardiovascular surgery, thoracicsurgery, head and neck surgery, pelvic surgery, and skin andsubcutaneous tissue procedure.

In one embodiment, the wounded tissue comprises a bleeding artery.

In one embodiment, the treatment comprises reducing bleeding in asubject, for example, a human subject.

According to another aspect of the present invention there is provided amethod of treating a wounded tissue in a subject, the method comprisingex vivo combining a liquid formulation comprising fibrinogen and/orfibrin with an oxidized cellulose backing, such as, and without beinglimited thereto, ORC backing, to form a wound dressing; and applying thewound dressing onto a wound, the application being carried out withinless than 20 seconds from the combining.

In some embodiments, application may be carried out within 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 5, 4, 3, 2, or 1 seconds, or evenimmediately upon the combining. In some embodiments, the applicationonto the wound is carried out after at least 2 to 5 seconds fromcombining, or in some embodiments, after at least 2 to 10 seconds fromcombining.

In one such embodiment, the backing has a wound facing side and theliquid formulation is combined, with at least the wound facing side or aportion thereof.

The invention provides use of a wound dressing for treating a wound in asubject in need by applying the dressing to the wound, the dressingcomprises fibrinogen and/or fibrin containing liquid formulation ex vivocombined with an oxidized cellulose backing, such as, and without beinglimited thereto, ORC backing, the applying being carried out within lessthan 20 seconds from combination of the liquid with the backing.

In one such embodiment, the backing or scaffold has a wound facing sideand the liquid formulation is combined at least partially with the woundfacing side.

In one such embodiment, the wounded tissue is a bleeding tissue.

In one such embodiment, the backing comprises ORC.

In one such embodiment, the backing is in the form of a knitted, wovenor non-woven fabric.

In one such embodiment, the fibrinogen and/or fibrin containingformulation further comprises thrombin.

In one such embodiment, the formulation comprises a two-componentformulation comprising: a thrombin component, such as, and without beinglimited thereto, a liquid thrombin component, and a fibrinogencomponent, such as, and without being limited thereto, a liquidfibrinogen component.

In one such embodiment, the formulation comprises a two-componentformulation comprising: a thrombin component, such as, and without beinglimited thereto, a liquid thrombin component, and a fibrin component,such as, and without being limited thereto, a liquid fibrin component.

In one such embodiment, the combining comprises one or more stepsselected from spraying, smearing, brushing, casting, printing, injectingand soaking the fibrinogen and/or fibrin containing liquid formulation

In one such embodiment, following the combining, the method comprisespressing the wound dressing against the wounded tissue.

In one such embodiment, the pressing is for a time sufficient to allowadherence of the wound dressing to at least a portion of the woundedtissue.

In one such embodiment, the use is for treating a wound during orfollowing a surgical procedure.

In one such embodiment, the surgical procedure comprises a procedureselected from abdominal surgery, cardiovascular surgery, thoracicsurgery, head and neck surgery, pelvic surgery, and skin andsubcutaneous tissue procedure.

In one such embodiment, the wounded tissue comprises a bleeding artery.

In one such embodiment, the use comprises reducing bleeding in asubject, for example, a human subject.

In another aspect, there is provided a kit for treating a wounded tissuein a subject, the kit comprising a first container comprising anoxidized cellulose (OC) backing, such as, and without being limitedthereto, ORC backing; and a second container comprising fibrinogenand/or fibrin containing formulation, and optionally instructions foruse according to the methods disclosed herein.

In one embodiment of the kit, the formulation is in a liquid or in afrozen form.

In a further embodiment of the kit, the backing is in the form of aknitted, woven or non-woven fabric.

In one embodiment, the kit comprises thrombin in a third container.

In one embodiment of the kit, the formulation comprises a two-componentformulation comprising: a thrombin component, such as, and without beinglimited thereto, a liquid thrombin component, and a fibrinogencomponent, such as, and without being limited thereto, a liquidfibrinogen component.

In one embodiment of the kit, the formulation comprises a two-componentformulation comprising: a thrombin component, such as, and without beinglimited thereto, a liquid thrombin component, and a fibrin component,such as, and without being limited thereto, a liquid fibrin component.

The following description, while describing the invention with referenceto the method of preparing the wound dressing, should be read as alsodescribing features of the other aspects of the disclosed invention,including the method of treatment, the wound dressing per se and the kitproduct.

For the purpose of producing the wound dressing disclosed herein, twomain components are utilized, the fibrinogen and/or fibrin containingliquid formulation and the OC backing.

The ex vivo combination of the fibrinogen and/or fibrin containingliquid formulation with an oxidized cellulose backing was found topermit adhesion to the bleeding wound, and thus address hemostasiswithout being depended by the condition of the wounded tissue, e.g.,severity of bleeding.

When referring to ex vivo, in the context of the present disclosure, itshould be interpreted as any location that is not in physical contactwith the wounded tissue.

The term “fibrinogen and/or fibrin containing liquid formulation” or“(fibrinogen-based sealant formulation”) is to be understood as a liquidformulation comprising fibrinogen and/or fibrin which may function as asealant. This liquid formulation is referred also a biological tissueadhesive formulation, such as formulation of: fibrin glue, fibrinsealant, fibrin adhesive, fibrin film, fibrin network, fibrin lattice,fibrin mesh, fibrin greed and fibrin gel. These formulations thus havingingredients that upon contact with the tissue or blood react tosubsequently reduce or stop bleeding and/or seal physiological leaks,e.g., of cerebrospinal fluids (CSF), lymph, bile, gastrointestinal (GI)content, air leak from lungs etc.

In the context of the present disclosure it is further to be understoodthat during storage the formulation ingredients do not interact witheach other and are essentially inert until they are brought into contactwith the member of the coagulation cascade. Once the member is activatedby the sealant components, clotting initiates and fibrin clot basedtissue adhesive is formed.

The fibrinogen component in the formulation may be purified and isolatedfibrinogen per se, but also may be biologically active component (BAC)of blood plasma comprising fibrinogen concentrated viral-inactivatedcryoprecipitate of human plasma comprising solution of blood plasmaderived proteins.

In some embodiments, the fibrinogen is provided as part of the BAC.There are several types of BAC. In some embodiments, BAC is abiologically active component that comprises tranexamic acid, as anantifibrinolytic agent.

In some other embodiments, BAC is a biologically active component thatdoes not contain tranexamic acid. This is considered a second-generationBAC and is referred to in the art as “BAC2”. During BAC2 preparation,plasminogen (the enzyme precursor of plasmin, which breaks downfibrinogen and fibrin) and/or plasmin is removed.

In one embodiment, BAC is BAC2, i.e. a biologically active componentthat lacks tranexamic acid.

BAC may be prepared as component A in the disclosure of EP 534 178. Forexample, component A therein is prepared from concentratedcryoprecipitate, and undergoes viral inaction by solvent detergenttreatment and pasteurization.

In some embodiments, BAC2 is a concentrated viral inactivatedcryoprecipitate comprising mainly fibrinogen and is plasminogen-depleted(the removal of plasminogen may be carried out as described in EP 1 390485). In view of removal of plasmin/plasminogen from thecryoprecipitate, there is no need to add anti-fibrinolytic agents, suchas, and without being limited thereto, tranexamic acid, aprotinin or thelike.

In some embodiments, fibrinogen is purified from an aluminum hydroxideprecipitate [a byproduct from the manufacture process of factor VIII(FVIII)] as disclosed in WO2013001524A1.

The BAC solution may further comprise stabilizers such as, and withoutbeing limited thereto, arginine, lysine or other sealant additives asknown in the art.

In some embodiments, BAC, optionally BAC2, may be derived fromcryoprecipitate (in particular concentrated cryoprecipitate).

Herein, unless otherwise stated, when referring to BAC it is to beunderstood as preferably, but not exclusively, referring to BAC2.

Examples of fibrinogen sources include, but are not limited to,recombinant fibrinogen, purified fibrinogen, and fibrinogen component.

In some embodiments, the fibrinogen and/or fibrin containing liquidformulation is provided as a part of a two-component formulation thatare to be blended together prior to use. Some such two componentsinclude a first, fibrinogen-containing component, and a second,thrombin-containing component.

In some embodiments, for the purpose of producing the wound dressingdisclosed herein, three main components are utilized: (i) a fibrinogencontaining component, (ii) a thrombin containing component, and (iii) anOC backing, such as, and without being limited thereto, ORC backing.

In some embodiments, for the purpose of producing the wound dressingdisclosed herein, a two-component fibrin sealant formulation is usedtogether with an OC backing, such as, and without being limited thereto,ORC backing. In some embodiments, the fibrin sealant formulationcomprises a fibrinogen containing component and a thrombin containingcomponent.

There are various commercially available fibrinogen-based sealantformulations.

For example, the sealant formulation is one comprising fibrinogen andthrombin, such as, and without being limited thereto, the FDA approvedsealant, marketed under the trademark EVICEL®, is a package containingone vial each of BAC2 (55-85 mg/ml BAC2) and thrombin (800-1200 IU/mlhuman thrombin) frozen solutions.

A further commercially available sealant is the FDA approved productmarketed under the tradename TISSEEL®. TISSEEL is a fibrin sealantprovided, as a two-component fibrin sealant comprising freeze-driedSealer Protein Concentrate (fibrinogen and aprotinin) and thrombin thatare reconstituted in fibrinolysis inhibitor solution and calciumchloride solution, respectively.

In some other embodiments, the formulation is a one/single componentsealant (glue) formulation comprising fibrinogen, calcium ion and factorXIa such as that described in PCT patent application No.PCT/IL2017/000006.

In some other embodiments, the fibrinogen and/or fibrin containingformulation is an aqueous formulation. When referring to an “aqueousformulation” it is to be understood to encompass a blend of ingredients,in liquid or frozen, that contains water molecules. In some embodiments,the aqueous formulation is in liquid form. When in liquid form, it is inaccordance with some embodiments that the liquid carrier is a bufferhaving an essentially neutral pH, e.g., pH 7.0±0.5.

In some embodiments, the formulation is frozen. Prior to use, theformulation may be thawed and thereby turn into liquid form at roomtemperature.

In yet some other embodiments, the formulation is in the form of apowder e.g., lyophilized and prior to use the powder is moistened withan aqueous solution.

As described hereinthroughout, in the context of the present disclosure,the oxidized cellulose (OC) backing also encompasses oxidizedregenerated cellulose (ORC) backing.

In some embodiments, the OC backing may be combined with othersubstances typically used as TAH, such as, any one or combination ofgelatin, collagen, chitin and chitosan.

In one embodiment, OC is present in the wound facing side whereas theother substances typically used as TAH such as, and without beinglimited thereto, any one or combination of gelatin, collagen, chitin andchitosan are present in the side that is not facing the wound.

In some embodiments, the OC backing is not combined with othersubstances typically used as TAH, such as, any one or combination ofgelatin, collagen, chitin and chitosan.

When referring to “backing” it is to be understood as meaning a physicalstructure, e.g. scaffold or patch made of at least OC. In someembodiments, the backing is in the form of a fabric. The fabric may bein a knitted, woven or non-woven form.

As used herein, the term “patch” relates to a piece of material used toprotect a part of the body, give shape to something or clean something,and is intended to include a pad, a cloth, a mesh, a dressing, a gauze,and the like.

As used herein, the term “pad” relates to a small piece of material thatcan be sewn or otherwise attached over a surface or wound.

As used herein, the term “cloth” relates to woven or felted fabric madefrom a fiber.

As used herein, the term “mesh” relates to a material made of a networkof connected strands of flexible materials such as wire or thread.

As used herein, the term “dressing” relates to a covering for a wound.

As used herein, the term “fabric” relates to a flexible materialcomprising a network of fibers produced by weaving, knitting,crocheting, knotting, felting or bonding.

As used herein, the term “gauze” relates to thin, loosely woven clothused for dressings and swabs or to any material made of an open,mesh-like weave.

The OC backing may provide a scaffold for the formation of the wounddressing.

In some embodiments, the “backing” is in the form of a powder, e.g. OCpowder.

Some of the most commonly used OC backing materials (topical hemostaticagents) belong to the family of agents marketed under the tradenameSURGICEL®. These are made from oxidized regenerated cellulose (ORC). TheSURGICEL® family of absorbable hemostats comprising four main productgroups, with all hemostatic wound dressings commercially available fromEthicon, Inc., Somerville, N.J., a Johnson & Johnson Company.

SURGICEL® Original hemostat is a white fabric with a pale-yellow castand a faint, caramel like aroma. This material is strong and may besutured or cut without fraying. Further, is a loose knit ORC fabric thatconforms rapidly to its immediate surroundings and is easy to managebecause it does not stick to surgical instruments and its size may beeasily trimmed. This allows the surgeon to hold the cellulose firmly inplace until all bleeding stops. The control of bleeding is essential andcritical in surgical procedures to minimize blood loss, to reducepost-surgical complications, and to shorten the duration of the surgeryin the operating room. Due to its biodegradability and its bactericidaland hemostatic properties, oxidized cellulose, as well as oxidizedregenerated cellulose have long been used as a topical hemostatic wounddressing in a variety of surgical procedures.

One member of the family is SURGICEL® NU-KNIT® absorbable hemostat,which is similar to SURGICEL® Original but has a denser knit and thus ahigher tensile strength, this material is particularly recommended foruse in trauma and transplant surgery as it may be wrapped or sutured inplace to control bleeding;

SURGICEL® FIBRILLAR™ absorbable hemostat product form has a layeredstructure that allows the surgeon to peel off and grasp with forceps anyamount of material needed to achieve hemostasis at a bleeding site, andtherefore, may be more convenient than the knitted form for hard toreach or irregularly shaped bleeding sites. It is particularlyrecommended for use in orthopedic/spine and neurological surgery;

SURGICEL® SNoW™ absorbable hemostat product form is a structurednon-woven fabric that may be more convenient than other forms forendoscopic use due to the structured, non-woven fabric, and is highlyadaptable and recommended in both open and minimally invasiveprocedures.

Typically, SURGICEL is a non-abrasive textured rayon fabric with a whiteto pale yellow cast. It has a Carboxyl Content of 18-21%. The knitstructure varies depending on the product:

SURGICEL Original—is a knitted material;

NUKNIT—is a further kind of a knitted material;

SURGICEL® FIBRILLAR™—Nonwoven;

SNoW—Light Nonwoven.

In some embodiments, the OC backing is INTERCEED™ is a non-abrasivetextured rayon fabric with a white to pale yellow cast. It has aCarboxyl Content of 12-18%. INTERCEED™ is a knitted material.

Typically, “nonwoven fabric” is used in the textile manufacturingindustry to denote fabrics, such as , and without being limited thereto,felt, which are neither woven nor knitted.

Typically, the term “nonwoven fabric” includes, but is not limited to,bonded fabrics, formed fabrics, or engineered fabrics, that aremanufactured by processes other than spinning, weaving or knitting. Thestructure of the nonwoven fabric is based on the arrangement of, forexample, staple fibers that are typically arranged more or lessrandomly. Notwithstanding, the raw materials used to manufacture thenonwoven fabric may be yams, scrims, netting, or filaments made byprocesses that include spinning, weaving or knitting (as disclosed inEP1802358A2).

In some embodiments, the OC backing has a face with which fibrinogenand/or fibrin is combined. This face is referred to as the “wound facingside” of the backing.

According to the present disclosure, the wound dressing is produced bycombining, at a location remote from the tissue to be treated with thedressing, the fibrinogen and/or fibrin-containing liquid formulationwith an oxidized cellulose (OC) backing, the combining includes at leastapplying or providing the fibrinogen and/or fibrin containingformulation to the wound facing side of the backing.

The combining of the OC backing and the fibrinogen and/or fibrincontaining formulation may be by any means known in the art. Theseinclude, without being limited thereto, application at least onto a sidedesignated as the wound facing side of the backing, by spraying,smearing, brushing, casting, printing, injecting, soaking, coating andany combination of same.

Without being bound by theory, it appears that the sealant componentscrosslink and adhere to the wound, creating a frail liquid-tight barrierwhich is supports by the TAH backing. The sealant components alsocrosslink within the porous OC fibers/TAH, mechanically locking the OCto the formed liquid-tight barrier.

The formation of the sealant (biological glue) occurs in a relativelyshort period of time subsequent to the combination of the liquidformulation and the OC backing. It has been found that there is a narrowtime window suitable for the combination of components and applicationonto the wound that provides a superior adhesiveness to the resultingdressing in comparison to other time windows.

In addition, the combination according to the invention was found to beadvantageous in cases of severe bleeding where application of thesealant directly onto the wound side could be washed away/diluted, asfurther addressed below.

As shown in the following non-limiting examples, it has been found thatthe application of the wound dressing onto the wound should be withinless than 20 seconds from the time of combination, i.e. with a minimalcross-linking time of less than 20 seconds.

This combination of the liquid formulation and the OC backing ex vivoprovides the wound dressing that is then applied onto the wound.

In some embodiments, the wound dressing is particularly advantageouswhen applied onto severely bleeding wounds, i.e. when the flow of bloodand/or other bodily fluids from the wound is intense.

In some embodiments, the placing of the ex vivo formed wound dressing iswithin any time point from the second of its production by combining thefibrinogen and/or fibrin-containing liquid formulation with the OCbacking, and until 19 seconds, at times, until 18, at times, until 17,at times, until 16, at times, until 15, at times, until 14, at times,until 13, at times, until 12, at times, until 11, at times, until 10, attimes, until 9, at times, until 8, at times, until 7, at times, until 6,at times, until 5, at times, until, 4, at times, or until 3 seconds fromthe moment of combination. In some embodiments, the application onto thewound is after at least 2-5 seconds from preparation, at times, after atleast 2-10 seconds from preparation.

Once the wound dressing is produced, it is applied onto the wound. Insome embodiment, this involves pressing it against the target/woundedtissue.

In some specific embodiments, the wounded tissue comprises a bleedingartery.

The wound dressing is pressed for a time sufficient to allow adherenceof the wound dressing to at least a portion of the wounded tissue. Inthis manner, the combined effect of the formed fibrin glue and theclotted blood of the subject can assist in controlling the bleeding ofthe wounded tissue.

In some embodiments, the wound dressing is pressed against the wounduntil bleeding is essentially stopped.

The methods disclosed herein and the produced wound dressing may be usedfor treating various animal tissues. In this context, the animal may bea human as well as a non-human animal.

In some embodiments, the animal is a mammal.

In some embodiments, the animal is a human subject.

Treatment with the wound dressing disclosed herein can be given duringany standard procedure, e.g. personal care (i.e. at home), in a clinic,during a surgical procedure etc.

There are many surgical procedures in which the wound dressing of thepresent disclosure can be used, such as, without being limited thereto,abdominal surgery, cardiovascular surgery, thoracic surgery, head andneck surgery, pelvic surgery, skin and subcutaneous tissue procedure.

To facilitate production of the wound dressing, the components thereof,namely, the fibrinogen containing formulation (be it a two-componentformulation or a single component formulation as described above) andthe OC backing can be provided as separate components within acommercial kit. To this end, the kit, e.g. including a label for use inwound treatment, contains a first container/package including thefibrinogen formulation, and a second container/package containing the OCbacking.

The fibrinogen containing formulation may be stored in liquid or frozenform such that, for example, it may be preserved for a long period whenit is in its frozen form and thawed just before use.

In some embodiments, the kit is provided with instructions for use ofthe OC backing in combination with the fibrinogen and/or fibrincontaining formulation for the ex vivo production of the wound dressingand application thereof onto the wound.

In some embodiments, the instructions comprise applying of the thusformed wound dressing at a time interval of less than 20 seconds (asdefined hereinabove) from the moment of the combination.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”,and their conjugates mean “including but not limited to”. The term“consisting of” means “including and limited to”. The term “consistingessentially of” means that the composition, method or structure mayinclude additional ingredients, steps and/or parts, but only if theadditional ingredients, steps and/or parts do not materially alter thebasic and novel characteristics of the claimed composition, method orstructure.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

In those instances where a convention analogous to “at least one of A,B, and C, etc.” is used, in general such a construction is intended inthe sense one having skill in the art would understand the convention(e.g., “a system having at least one of A, B, and C” would include butnot be limited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). It will be further understood by those within the artthat virtually any disjunctive word and/or phrase presenting two or morealternative terms, whether in the description, claims, or drawings,should be understood to contemplate the possibilities of including oneof the terms, either of the terms, or both terms. For example, thephrase “A or B” will be understood to include the possibilities of “A”or “B” or “A and B.”

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

In the following non-limiting Example, cross linking time and adhesionintensity of the wound dressing disclosed herein was examined in anex-vivo model.

Example 1 Adhesion Intensity in an Ex-Vivo Model Materials:

Porcine skin tissue with exposed dermis was received from Lahav contractresearch organization (C.R.O.) and delivered in chilled containers (4 °C.).

For the application of the fibrin sealant, the EVICEL® airless sprayaccessory tip was assembled on top of a 5 ml EVICEL® device.

EVICEL® is a package containing one vial each of BAC2 (55-85 mg/ml BAC2)and thrombin (800-1200 IU/ml human thrombin).

In exemplary procedures (including Example 2), the BAC2 component andthe thrombin component are combined at ratio of 1:1 (v/v) (and aretherefore diluted accordingly.)

When EVICEL® was applied, either directly on the tissue or on SURGICEL®,lml of the fibrin sealant was used.

When SURGICEL® was applied, SURGICEL NU-KNIT® was cut to a 5.08 cm X5.08 cm patch size to be used either as a control or with combination ofEVICEL®.

Adhesion Intensity Ex-Vivo Model:

Porcine skin tissue was used to evaluate the adhesion intensity of awound dressing prepared from a fibrin sealant and an ORC patch andapplied to tissue in accordance with the Method of the presentdisclosure vis-à-vis the reference method (as described below).

Method of the present disclosure: fibrin sealant EVICEL® was applied onthe ORC patch SURGICEL NU-KNIT® and the combined product was applied,jointly, onto the porcine skin tissue.

Reference Method: the fibrin sealant EVICEL® was applied on the porcineskin tissue followed by the application of the ORC patch SURGICELNU-KNIT® on top of EVICEL®.

Also, in this study, the effect of EVICEL® crosslinking time (“curingtime”) on the adhesion intensity was examined. To this end, once thefibrin sealant was applied, either on SURGICEL® or directly onto thetissue, the adhesion intensity at different “curing times” wasdetermine.

Method:

Porcine skin tissue was placed dermis side facing up and hydrated usingsaline (0.9%) soaked gauze to keep the tissue moist when necessary. 1 mlof fibrin sealant EVICEL® was applied in combination with a 5.08 cm×5.08cm patch of SURGICEL NU-KNIT® on the dermis. EVICEL® was either applieddirectly on the tissue followed by the application of SURGICEL®, or,EVICEL® was applied on SURGICEL® followed by their joined application tothe tissue.

To examine the effect of EVICEL® crosslinking time (“curing time”) onthe adhesion intensity, Fibrin sealant was applied, either on SURGICEL®or on the tissue, and two “curing times” were taken (immediately, i.e.at time of around 0 seconds and at 20 seconds from the application ofEVICEL® to the tissue or to the SURGICEL® pad).

At the specified time, the ORC patch (with or without the fibrinsealant) was applied onto the skin. After two minutes in situ, thedressing (patch with fibrin sealant) was pulled from the tissue usingforceps and the adhesion intensity was manually assessed on a scale of0-5; whereby no adhesion was given a score of 0 and intensive adhesionwas given a score of 5. As a control, the adhesion force of SURGICEL®without EVICEL® was also measured.

Results:

The fibrin sealant (fibrin glue) was either applied directly on thetissue followed by the application of the ORC patch (Reference Method),or, the fibrin sealant (fibrin glue) was applied on the ORC patchfollowed by the coated patch application on the tissue (Method disclosedherein).

Different crosslinking/curing times were taken at which the patch'sadhesion intensity to the tissue was assessed on a scale of 0-5. As acontrol, the adhesion force of SURGICEL® without EVICEL® was alsomeasured.

The results are shown in FIG. 1. Specifically, FIG. 1 presents, in theform of a bar graph, the strength required in order to remove the patch,namely the wound dressing, from the tissue.

The most left bars identified as “immediately” show that when thecombined patch/fibrin sealant is applied by either method to the tissue,almost the same amount of force is required in order to remove/peal thepatch from the tissue. However, as also shown, it is essential that whenthe combination takes place ex vivo, the application of the combinedpatch has to be in less than 20 seconds, otherwise the intensity ofadhesion is reduced.

In other words, the results in FIG. 1 demonstrate no statisticalsignificant between the application techniques when they are immediatelyapplied. This demonstrate how the novel technique is at least anequivalent method with regards to adherence strength. However,statistical significant can be found when the patch including thecombined patch/fibrin sealant application is carried out 20 second afterthe fibrin sealant application. This demonstrates the importance oftiming in the application method.

Thus, while there is an advantage of combining the fibrin formulationwith the backing ex vivo (there is no washing off of the formulation bythe bleeding wound, e.g. in cases of severe bleeding), this has to bedone in less than 20 seconds so as to retain the strength/adherence ofthe dressing.

Example 2 Adhesion Intensity in an In-Vivo Model

The in vivo efficacy of Fibrin sealant applied on an ORC patch SURGICELNU-KNIT® was examined next.

The test articles were prepared by the application of fibrin sealant 1ml to an ORC patch (5.08 cm×5.08 cm). Next, various crosslinking times(crosslinking time being defined as time from application of the fibrinsealant to the ORC until application of the test article to the defectsite) were allowed for the applied fibrin sealant following the testarticle application to the defect site. The examined crosslinking timeswere: around 0 seconds (immediately), after 10 seconds and after 20seconds of crosslinking time.

The animal procedure used is based on the procedure previously reportedin MacDonald et al. 2017, Medical Devices: Evidence and Research 10:273-279, with the following modifications:

-   -   a disposable biopsy punch of 8 mm with a stop set to 2 mm was        used;    -   a tamponade was applied for 30 seconds after the test article        application and the presence of free-flowing blood was monitored        for 2 minutes;

Briefly, a ventral midline abdominal incision was performed in a porcineand the cranial portion of the midline incision was extended to improveexposure of the liver. The liver was positioned as necessary to maximizetesting surface availability. The abdominal organs were kept moist withsaline and saline-soaked laparotomy sponges throughout the procedure.The liver parenchymal defects were created on the diaphragmatic surfaceof accessible areas of the left, right, and quadrate lobes using adisposable 8 mm biopsy punch with a depth stop set to 2 mm. The coreportion of the biopsy was grasped and sharply dissected free from theunderlying surface causing mild to moderate hemorrhage.

The defect site was allowed to bleed for several seconds prior toproduct application to allow for characterization of the resultinghemorrhage. The trial site was blotted with gauze and then one of thetest articles was applied. For the purposes of this study, effectivehemostasis was defined as the cessation of free flow bleeding. Pinpointor petechial bleeding that appeared but did not grow was not consideredto be free flow bleeding. The test article was prepared and then appliedonto the defect (bleeding site) manually. Tamponade was applied for 30seconds using digital pressure on a gauze. Following the initial 30seconds of tamponade, the dressing was removed. If hemostasis was notachieved after 120 seconds, the test article was determined as unable toachieve effective hemostasis. The efficacy results of the fibrin sealantapplied (EVICEL ®) on an ORC patch (SURGICEL® pad), following theirapplication to the defect site with different crosslinking times aredisplayed in Table 1.

TABLE 1 in vivo hemostasis efficacy of fibrin sealant applied to an ORCpatch with different crosslinking times Crosslinking times Hemostasisachieved (Yes/No) Around 0 seconds (immediate Yes application) 10seconds Yes 20 seconds No

The results demonstrate that the effective hemostasis can be achieved byfibrin sealant application to ORC followed by their combined applicationto the defect site, but only if the allowed crosslinking time is below20 seconds in this porcine model.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1-5.
 6. A wound dressing comprising an ex vivo formed combination of aliquid formulation comprising fibrinogen and/or fibrin and an oxidizedcellulose (OC) backing.
 7. The wound dressing of claim 6, wherein saidliquid formulation is combined at least partially with a wound facingside of said wound dressing.
 8. The wound dressing of claim 6, whereinthe OC backing comprises oxidized regenerated cellulose (ORC).
 9. Thewound dressing of claim 6, wherein said liquid formulation furthercomprises thrombin. 10-17.
 18. A kit for treating a wounded tissue in asubject comprising: a first container comprising an oxidized cellulose(OC) backing; a second container comprising a formulation comprisingfibrinogen and/or fibrin; and optionally, instructions for use.
 19. Thekit of claim 18, wherein said formulation is in a liquid or in a frozenform.
 20. The kit of claim 18, further comprising thrombin within athird container.